Methods for treating inflammation, inflammatory diseases, arthritis and stroke using pADPRT inhibitors

ABSTRACT

The present invention is directed to a method for treating inflammation or inflammatory disease, bacterial infection, arthritis and stroke in an animal or mammal, which comprises the steps of administering an effective amount of a pADPRT inhibitory compound to said animal or mammal.

The present application is a continuation-in-part of U.S. Ser. No.08/855,616 filed on May 13, 1997, now U.S. Pat. No. 5,908,861.

The present invention relates to methods for treating inflammation andinflammatory diseases, arthritis, and stroke in animals. The inventionalso relates to methods for treating animals having toxicity resultingfrom infestation by lipopolysaccharides. These methods involve the useof therapeutically effective amounts of pADPRT inhibitory compounds.

BACKGROUND OF THE INVENTION

The use of pADPRT inhibitory compounds have been reported for treatingcancer and viral infections. Examples of these methods are described inU.S. Pat. Nos. 5,464,871, 5,473,074; 5,482,975, 5,484,951; 5,516,941,and 5,583,155, the disclosures of which are incorporated herein byreference.

In the published literature, 5-iodo-6-amino-1,2-benzopyrone (INH₂BP), anovel inhibitor of the nuclear enzyme poly-ADP ribose polymerase(PADPRT) has recently been shown to inhibit in vivo tumorigenicity in aHa-ras transfected endothelial cell line (Bauer et al., Int. J. Oncol.8:239-252 (1995) and Bauer et al., Biochimie 77:347-377 (1995)).Treatment with INH₂BP has also resulted in changes in topoisomerase Iand II and MAP kinase activity Based on the effects observed, ahypothesis regarding the potential use of INH₂BP in the therapy ofcancer has been put forward.

Malignant growth and inflammatory processes may feature the activationof certain common cellular signal transduction pathways, e.g., MAPkinase (Kyriakis et al., J. Biol. Chem. 271:24313-24316 (1996) andFerrell, TIBS 21:460-466 (1996)). Chronic inflammation frequently leadsto carcinogenic transformation, as demonstrated, for example, in thecase of the intestine. In our study, the production of multipleproinflammatory mediators was induced by bacterial lipopolysaccharide(endotoxin, LPS). LPS is known to induce a multitude of cellularreactions and triggers a systemic inflammatory response. LPS-inducedpro-inflammatory mediators include tumor necrosis factor alpha (TNF),interleukin-1, interferon-gamma, whereas antiinflammatory mediatorsinclude interleukin-10 (IL-10) and interleukin-13 (Deltenre et al., ActaGastroenterol Belg. 58:193-200 (1995), Beutler, J. Invest. Med.42:227-35 (1995), Liles et al., J. Infect Dis. 172:1573-80 (1995), andGiroir, Critical Car. Med. 21:780-9 (1993)). As a consequence of theproduction of these inflammatory cytokines, LPS initiates the productionof inflammatory free radicals (oxygen-centered, such as superoxide, andnitrogen-centered radicals, such as nitric oxide (NO) and ofprostaglandins (Nathan, FASEB J. 6:3051-3064 (1992), Vane, Proc. Roy.Soc. Lond B 343:225-246 (1993), and Szabo, New Horizons 3:3-32 (1995)).The production of NO in inflammation is due to the expression of adistinct isoform of NO synthase (iNOS), while the production ofinflammatory cytokines is explained by the expression of a distinctisoform of cyclooxygenase (cyclooxygenase-2, COX-2), iNOS, COX-2, aswell as other pro-inflammatory cytokines and free radicals which play animportant role in the LPS-induced inflammatory response. Moreover, NO(or its toxic byproduct, peroxynitrite), has been implicated as a keymediator leading to the transformation of the inflammatory response intoa carcinogenic process (Bartsch et al., Pharmacogenetics 2:272-7 (1994),Liu et al., Carcinogenesis 15:2875-7 (1992) and Ohshima et al., MutationRes. 305:253-64 (1994)).

There are a multitude of intracellular processes which precede theproduction of proinflammatory mediators. Activation of tyrosine kinases(Levitzki, A., Eur. J. Biochem. 226:1-13 (1994), Novogrodsky et al.,Science 264:1319-22 (1994), Marczin et al., Am. J. Physiol.265:H1014-1018 (1993)), mitogen-activated protein kinase (MAP kinase,Matsuda et al., J. Leukocyte Biol. 56:548-53 (1994), L'Allemain, Progr.Growth Factor Res. 5:291-334 (1994), and Cowley et al., Cells 77:841-52(1994)); and the nuclear factor kappa B (NF-kB) pathway (Baeuerle etal., Ann. Rev. Immunol. 12:141-79 (1994), Schreck et al., Free RadicalRes. Comm. 17:221-37 (1992) and Muller et al., Immunobiol. 187:233-56(1993)) are recognized as important factors in the inflammatory responseand contribute to the expression or production of inflammatorymediators.

SUMMARY OF THE INVENTION

One aspect of the invention is a method for treating inflammation orinflammatory disease in an animal or mammal, which comprises the stepsof administering an effective amount of a pADPRT inhibitory compound.

Another aspect of the invention is a method for treating inflammation orinflammatory disease in an animal or mammal, which comprises the stepsof administering an effective amount of a pADPRT inhibitory compoundwherein the pADPRT inhibitory compound is selected from the groupconsisting of:

a compound having the formula:

wherein R₁, R₂, R₃, R₄, R₅, and R₆ are each selected from the groupconsisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl orphenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, andonly one of R₁, R₂, R₃, R₄, R₅, and R₆ is selected from the groupconsisting of amino, nitroso or nitro; a compound having the formula:

wherein R₁, R₂, R₃, R₄, and R₅ are each selected from the groupconsisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl orphenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, andonly one of R₁, R₂, R₃, R₄, and R₅ is selected from the group consistingof amino, nitroso or nitro; and a compound having the formula:

wherein R₁, R₂, R₃, R₄, and R₅ are each selected from the groupconsisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl orphenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, andonly one of R₁, R₂, R₃, R₄, and R₅ is amino. Preferred pADPRT compoundsinclude: 6-amino-1,2-benzopyrone, 3-nitrosobenzamide,5-amino-1(2H)-isoquinolinone, 7-amino-1(2H)-isoquinolinone, and8-amino-1(2H)-isoquinolinone. Particularly preferred is the compound5-iodo-6-amino-1,2-benzopyrone.

Still another aspect of the invention is a method of treating arthritisin an animal comprising the step of administering an effective amount ofor a pADPRT inhibitory compound wherein the compound has the structuralformula noted above as compounds I, II or III. Especially preferred isthe compound 5-iodo-6-amino-1,2-benzopyrone.

Still another aspect of the invention is a method of treatingcerebrovascular accidents such as stroke in an animal comprising thestep of administering an effective amount of or a pADPRT inhibitorycompound wherein the compound has the structural formula noted above ascompounds I, II or III. Especially preferred is the compound5-iodo-6-amino-1,2-benzopyrone.

The pADPRT inhibitory compounds of the invention may be prepared by themethods described in U.S. Pat. Nos. 5,464,871, 5,473,074; 5,482,975,5,484,951, 5,516,941, and 5,583,155, the disclosures of which areincorporated herein by reference.

The preferred compounds for use in the methods of the invention includethose where the halo group is iodo, and one of the R groups is amino.Also, it has been found that the pADPRT inhibitory activity is stronglyexhibited when an iodo moiety is adjacent to an amino moiety. In anyevent, the compounds to be used in the methods of the invention shouldhave pADPRT inhibitory activity. The compounds may be used as is, orpreferably in combination with a pharmaceutically acceptable acidaddition salt or other suitable pharmaceutical carrier known in the art.

Those of skill in the art will readily understand that the pathologiesand disease states expressly stated herein are not intended to belimiting. Rather, the compounds of the present invention may be used totreat any disease which features an inflammatory response. That is, thecompounds of the present invention have pADPRT inhibitory activity andmay be effectively administered to ameliorate any disease state which ismediated all or in part by pADPRT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes the effect of INH₂BP on the development ofcarrageenan-induced paw edema. Data show paw volumes at 1-4 h aftercarrageenan injection (means ±S.E.M., n=6 animals in each group). Therewas a significant increase in the paw volume from hour 1 (p<0.01), andthere was a significant inhibition of the development of paw edema ofINH₂BP at 1-4 hours (**p<0.02).

FIG. 2 describes the effect of INH₂BP on the onset of collagen-inducedarthritis. The percentage of arthritic mice (mice showing clinicalscores of arthritis >1) are represented. The arrow at 21 days representsthe time of the second collagen immunization, the horizontal bar fromday 25 represents the time of the start of treatment with INH₂BP (N=6)or VEHICLE (N-10).

FIG. 3 describes the effect of INH₂BP on the severity ofcollagen-induced arthritis. The median arthritic score duringcollagen-induced arthritis is represented. The arrow at 21 daysrepresents the time of the second collagen immunization, the horizontalbar from day 25 represents the time of the start of treatment withINH₂BP (n-6) or vehicle (n=10). There was a significant increase in thearthritic score from day 26 (Ip<0.01), and there was a significantsuppression of the arthritic score by INH₂BP between days 26-35(#p<0.05).

FIG. 4 describes the representative histology of paw (a) control; (b)arthritic; (c) INH₂BP treatment+arthritis. Magnification: ×20.

FIG. 5 represents the suppression of the induction of iNOS in consciousrats treated with INH₂BP. iNOS activity in lung homogenates (a) andplasma nitrite-nitrate concentrations (b) in control rats (c), in ratsinjected with INH₂BP (INH₂BP); in rats injected with LPS (15 mg/kg i.p.for 6 h); and the effect of treatment with INH₂BP (10 mg/kg i.p.), whengiven 10 min. prior to LPS (INH₂BP+LPS) or at 2 hours after LPS(LPS+INH₂BP). ** represents a significant effect of LPS when compared tocontrols (p<0.01); ## represents significant inhibition by the pADPRTinhibitor (p<0.01); n=4-5.

FIG. 6 represents the effect of INH₂BP (10 mg/kg i.p.) on theLPS-induced TNF, IL-10 and IL-6 responses in mice, at 90 min. after LPSadministration (4 mg/kg i.p.). ## represents a significant effect of LPSwhen compared to controls (p<0.01); ## represents significantaugmentation of the response by INH₂BP (p<0.01); n=4-5.

FIG. 7 represents the improved survival in INH₂BP treated mice subjectedto endotoxin shock: effect of INH₂BP pretreatment (0.3-10 mg/kg) onendotoxin-induced (120 mg/kg i.p.) mortality in mice; n=7-8 animals ineach group.

FIG. 8 (a) MAP kinase activity in RAW 264.7 cells treated with vehicleor LPS (10 μg/ml) for 24 hours in the presence or absence of 100 μM PD98059 or 150 μM INH₂BP. Data represent values obtained in a typicalexperiment. Similar results were seen on 3 different experimental days.(b) Representative in gel MAP kinase assay in RAW 264.7 cells at 24hours after vehicle or LPS treatment in the presence or absence of 150μM INH₂BP. Lanes 1-4 represent the following groups, respectively: 1.vehicle-treated control; 2. LPS treatment; 3. vehicle treatment in thepresence of 150 μM INH₂BP; 4: LPS treatment in the presence of 150 μMINH₂BP.

FIG. 9 describes how inhibition of pADPRT with INH₂BP does not alter thenuclear translocation of NF-_(K)B Western blot of nuclear extracts ofcontrol J74 cells and cells at 90 minutes after LPS treatment in thepresence or absence of INH₂BP (100 μM).

FIG. 10 describes the effect of INH₂BP on the development ofcarrageenan-induced paw edema. Data show paw volumes at 1-4 hours aftercarrageenan injection (means ±S.E.M., n=6 animals in each group). Therewas a significant increase in the paw volume from hour 1 (p<0.01), andthere was a significant inhibition of the development of paw edema ofINH₂BP at 1-4 hours (**p<0.02).

FIG. 11 describes the effect of INH₂BP on the onset of collagen-inducedarthritis. The percentage of arthritic mice (mice showing clinicalscores of arthritis >1) are represented. The arrow at 21 days representsthe time of the second collagen immunization, the horizontal bar fromday 25 represents the time of the start of treatment with INH₂BP (N=6)or VEHICLE (N-10).

FIG. 12 describes the effect of INH₂BP on the severity ofcollagen-induced arthritis. Median arthritic score duringcollagen-induced arthritis is represented. The arrow at 21 daysrepresents the time of the second collagen immunization. The horizontalbar from day 25 represents the time of the start of treatment withINH₂BP (n-6) or vehicle (n=10). There was a significant increase in thearthritic score from day 26 (Ip<0.01), and there was a significantsuppression of the arthritic score by INH₂BP between days 26-35(#p<0.05).

FIG. 13 describes the effect of peroxynitrite (50-1000 μM) onmitochondrial respiration (a) and PARS activity (b) in PARS^(+/+) andPARS^(−/−) fibroblasts (i.e. the effect of INH₂BP (100 μM) in both celltypes). *^(,)** represents significant effects of peroxynitrite inPARS^(+/+) cells when compared to unstimulated controls (p<0.05, p<0.01,respectively). ^(#,##) represents significant effect of INH₂BP in thepresence of peroxynitrite (p<0.05, p<0.01, respectively); n=6-9.

FIG. 14 describes the effect of INH₂BP and 3-aminobenzamide on theoxidation of dihydrorhodamine 123 by hydrogen peroxide (top panel) orperoxynitrite (bottom panel). *^(,)** represents significant inhibitionof dihydrorhodamine oxidation by 3-aminobenzamide in response tohydrogen peroxide (p<0.05, p<0.01, respectively; n=6).

FIG. 15 represents (a) concentrations of nitrite (solid bars) or nitriteand nitrate (hatched bars), in PARS^(+/+) and PARS^(−/−) fibroblastsimmunostimulated with LPS/IFN for 24 hours; effect of INH₂BP (50-100μM). ** represents significant effects of immunostimulation whencompared to unstimulated controls (p<0.01), ^(##) represents significanteffect of INH₂BP in the PARS^(+/+) cells (p<0.01); n=6-9. (b) Comparisonof the level of iNOS mRNA (at 2, 8, 12 and 24 h) and iNOS protein (at 12h) expression in PARS^(+/+) and PARS^(−/−) cells in response tostimulation with IFN and LPS. Representative blots of n=3-4 experimentsare shown.

FIG. 16 represents nitrotyrosine immunostaining in (a) the paw of acontrol mouse and (b) the paw of a mouse at 35 days aftercollagen-induced arthritis. Part (c) represents nitrotyrosine Westernblots from paw extracts of control mice (C) and mice after 35 days ofcollagen-induced arthritis (A) are shown. Note the marked increase innitrotyrosine staining in the paws in arthritis. Also, note theincreased tyrosine nitration of several proteins (indicated witharrowheads): three proteins (Mw: approx. 60-80 kDa), and a low molecularweight protein or protein fragment (Mw: approx. 10 kDa). Representativepictures or gels of n=3 experiments are shown. For parts (a) and (b),magnification: ×40.

FIG. 17 represents the (A) Effect of INH₂BP on the onset ofcollagen-induced arthritis. The percentage of arthritic mice (miceshowing clinical scores of arthritis >1) are represented. (B) representsthe effect of INH₂BP on the severity of collagen-induced arthritis.Median arthritic score during collagen-induced arthritis is represented.The arrow at 21 days represents the time of the second collagenimmunization. The horizontal bar from day 25 represents the time of thestart of treatment with INH₂BP (n=6) or vehicle (control; C) (n=10).There was a significant increase in the arthritic score from day 26(*p<0.01), and there was a significant suppression of the arthriticscore by INH₂BP between days 26-35 (#p<0.05).

FIG. 18 depicts a representative histology of the paw (a) control; (b)arthritic; (c) INH₂BP treatment+arthritis. Note the reduction in thedegree of mononuclear cell infiltration in the paws of theINH₂BP-treated arthritic animals. Magnification is ×20.

FIG. 19 represents (A) INH₂BP prevention of PARS activation (top panel),the decrease in mitochondrial respiration, (middle panel) and LDHrelease (bottom panel) in peroxynitrite (500 μM) stimulated C6 cells.(B) Reduction in infarct size by INH₂BP in stroke. Brain infarct volume(top panel) and brain infarct area (bottom panel) in vehicle-injectedmice (diamonds) and mice treated with 10 or 30 mg kg⁻¹ INH₂BP ispresented. Data are presented as mean ±SE (n=6-12 and 8-11 per group inpanels a and b, respectively) *p<0.05 or **p<0.01 vs vehicle.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Definitions as used Herein

“Inflammatory” diseases refers to diseases or conditions where there isan inflammation of the body tissue. Such diseases include for example,Chron's disease, Barrett's disease, arthritis, multiple sclerosis,cardiomyopathic disease, colitis, infectious meningitis, encephalitis,and the like. “ADPRT” refers to adenosinediphosphoribose transferase andis also known as poly(ADP-ribose) polymerase (EC 2.4.99), a specificDNA-binding nuclear protein of eucaryotes that catalyzes thepolymerization of ADP-ribose. The enzymatic process is dependent on DNA.The term is synonymous with the term “PARS” or poly (ADP-ribose)synthetase in the literature. That is, the terms are used interchageablyherein and in the literature as is readily appreciated by those skilledin the art.

“Alkyl” refers to saturated or unsaturated branched or straight chainhydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

“Alkoxy” refers to the radical —O-alkyl. Typical alkoxy radicals aremethoxy, ethoxy, propoxy, butoxy and pentoxy and the like.

“Arthritis” refers to any condition affecting the joints of the skeletalsystem including degenerative conditions and autoimmune conditions. Suchconditions may generally be characterized by an influx of inflammatorycells.

“Cycloalkyl” refers to saturated monocyclic hydrocarbon radicalcontaining 3-8 carbon atoms such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

“Substituted phenyl” refers to all possible isomeric phenyl radicalssuch as mono or disubstituted with a substituent selected from the groupconsisting of alkyl, alkoxy, hydroxy, or halo.

The pADPRT inhibitory compounds of the invention (notably compoundsdefined above as compounds I, II or III) are potent, specific andnon-toxic anti-inflammatory compounds, that can be used for conditionsand diseases typically known for inflammation, such as arthritis,Chron's disease, Barrett's disease, and the like. Also, these compoundsare useful in the treatment of conditions associated with endotoxinpoisoning, especially those associated with gram negative bacteriainfections. Moreover, the compounds are useful for treating arthritisand stroke. The preferred compounds of the present invention such as5-iodo-6-amino-1,2-benzopyrone are especially useful in that they havevery low, if any, toxicity.

In practice, the compounds of the invention, or their pharmaceuticallyacceptable salts, may be administered in amounts that are sufficient toinhibit inflammatory conditions or disease and/or prevent thedevelopment of inflammation or inflammatory disease and may be used inthe pharmaceutical form most suitable for such purposes. Likewise, thecompounds of the invention may be administered in amounts which will besufficient to inhibit arthritis and pathology that is a sequelae ofcerebrovascular accident.

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for therapeutic agents.These methods include systemic or local administration such as oral,parenteral, transdermal, subcutaneous, or topical administration modes.The preferred method of administration of these drugs is oral. However,in some instances it may be necessary to administer the composition inparenteral form.

Depending on the intended mode, the compositions may be in the solid,semi-solid or liquid dosage form, such as, for example, injectables,tablets, suppositories, pills, time-release capsules, powders, liquids,suspensions, or the like, preferably in unit dosages. The compositionswill include an effective amount of one or more active pADPRT inhibitorycompounds or possibly the pharmaceutically acceptable salts thereof. Inaddition, it may include any conventional pharmaceutical excipients andother medicinal or pharmaceutical drugs or agents, carriers, adjuvants,diluents, etc., as customary in the pharmaceutical sciences.

For solid compositions such excipients may include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the likemay be used. The active pADPRT inhibitory compound defined above may bealso formulated as suppositories using, for example, polyalkyleneglycols, for example, propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. the active compound in apharmaceutical solution such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form theinjectable solution or suspension.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances suchas, for example, sodium acetate, triethanolamine oleate, etc.

Also, if desired, the pharmaceutical composition to be administered maycontain liposomal formulations comprising a phospholipid, a negativelycharged phopholipid and a compound selected from cholesterol, a fattyacid ester of cholesterol or an unsaturated fatty acid.

Parental injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions or solid forms suitable for dissolving in liquid prior toinjection.

A more recently devised approach for parenteral administration employsthe implantation of slow-release or sustained-released systems, whichassures that a constant level of dosage is maintained, according to U.S.Pat. No. 3,710,795, the disclosure of which is incorporated herein byreference.

Any of the above pharmaceutical compositions may contain 0.1-99%,preferably 1-70% of the active pADPRT inhibitory compounds, especiallythe halo-C-amino, nitroso or nitro compounds of the formulae I, II orIII, above as active ingredients.

The compounds of the present invention including5-iodo-6-amino-1,2-benzopyrone (INH₂BP) have been shown to regulate avariety of cellular signal transduction pathways and to abrogate in vivotumorigenicity by a Ha-ras transfected endothelial cell line. As oneaspect of the present invention demonstrates the effect of pADPRTinhibitory compounds such as INH₂BP on the activation by endotoxin(bacterial lipopolysaccharide, LPS) on the production of theinflammatory mediators tumor necrosis factor alpha (TNF), interleukin-10(IL-10) and interleukin-6 (IL-6), nitric oxide (NO) and prostaglandinsin vitro and in vivo. In addition, the pADPRT inhibitory effects of thecompounds of the present invention such as INH₂BP on the activation ofmitogen-activated protein kinase (MAP kinase) and nuclear factor kB(NF-kB) in vitro.

In cultured J774 and RAW 264.7 macrophages, LPS induced the productionof prostaglandin metabolites, the release of TNF and the expression ofthe inducible isoform of NO synthase (iNOS). The production ofprostaglandins and of NO are inhibited by INH₂BP in a dose-dependentmanner, while the short-term release of TNF-alpha is unaffected. INH₂BPmarkedly suppresses LPS-mediated luciferase activity in RAW cellstransiently transfected with a full length (−1592 bp) murine macrophageiNOS promoter-luciferase construct, but not in a deletional constructconsisting of −367 bp. In vivo, INH₂BP pretreatment inhibits theinduction of iNOS by LPS in rats, does not affect the LPS-induced TNFand IL-6 response, but enhances LPS-induced IL-10 production. INH₂BPpretreatment markedly improves the survival of mice in a lethal model ofendotoxin shock. These results demonstrate that pADPRT inhibitorycompounds such as INH₂BP have potent anti-inflammatory action in vitroand in vivo.

The following examples serve to illustrate the invention. They shouldnot be construed as narrowing it, or limiting its scope.

EXAMPLE 1

Cell Culture.

The mouse macrophage cell lines J774 and RAW 264.7 were cultured inDulbecco's modified Eagle's medium (DMEM) as described (Szabo et al.,Proc. Natl. Acad. Sci. U.S.A. 93:1753-1758 (1996) and Zingarelli et al.,J. Immunol. 156:350-358 (1996)). In separate studies, peritonealmacrophages were obtained from male Wistar rats and cultured in vitrofor 24 hours in the absence or presence of LPS and with or withoutINH₂BP. Rats were sacrificed and peritoneal macrophages taken andcultured in DMEM. Cells were treated with E. Coli LPS (10 mg/ml) or LPSand INF (50 u/ML) for various times, in the presence or absence ofvarious concentrations (1-150 mM) INH₂BP or other pharmacologicalinhibitors.

MAP kinase related assays.

Raw cells were washed in PBS and collected and lysed using 100 ml oflysis buffer per million cells. (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.4 MNaCl, 0.1 mM NaVO₃, 50 mM KF, 1 mM EGTA, 2 mM PMSF, 25 nM okadaic acid,1 mg/mL of each leupeptin, aprotinine, arnastatine and antipaine). Lysiswas carried out for 20 minutes on ice followed by a 14 min.centrifugation at 13000 rpm in an Eppendorf centrifuge. Supernatantswere saved and their protein content was assayed using the Bio-Rad dyeassay.

In gel MAP kinase assay.

Protein samples (50 mg/lane) were electrophoresed in a 10% SDS-PAGE gelcontaining immobilized myelin basic protein (MBP, 250 mg/mL gel). Afterelectrophoresis, the gel was washed once with 50 mM TRIS-HCl pH 7.7buffer (25 mL, 20 min.), followed by two 30 min. incubations with thesame buffer containing 25% i-propanol. The gel was then washed once withthe Tris-HCl buffer and soaked into a solution of 50 mM Tris-HCl pH 7.7,7 mM 2-mercaptoethanol, 5 M guanidine hydrochloride (50 mL) for an hour,changing the incubating solution at 30 minutes. The proteins were thenrenatured by incubating the gel in five changes of a solution of 50 mMTRIS-HCl pH 7.7, 7 mM 2-mercaptoethanol, 0.04% NP-40 over a 16 hourperiod of time. The gel was then washed twice and pre-incubated for halfan hour in a solution containing 50 mM TRIS-HCl pH 7.7, 5 mM MgCl₂, 7 mM2-mercaptoethanol. The final incubation was carried out in the samesolution supplemented with 10 mM of [³²P]-dATP (50 mCi/assay) for anhour. At the end of the incubation, the gel was washed free of unboundradioactivity using 3×25 mL of 10% TCA and 3×25 mL of 10% acetic acid,dried and auto-radiographed (Sasaki et al., Biochem. J. 311:829-34(1995)).

MAP kinase Western blotting.

One hundred mg of cell extract proteins were loaded onto a 10% SDS-PAGEgel, electrophoresed, transblotted onto nitrocellulose membrane andimmunoprobed. The first antibody (anti-MAP kinase) was from UBI, thesecond antibody was alkaline phosphatase labeled and from NEN Biolabs.Detection was by enhanced chemiluminesence (Bauer et al., Int. J. Oncol.8:239-252 (1995)).

Preparation of nuclear extracts and NF-kB Western blotting.

Cells were treated with LPS in the presence and absence of INH₂BP for 90minutes. Mininuclear extracts were prepared as described (Hassanain etal., Anal. Biochem. 213:162-7 (1993)). Briefly, cells were scraped,briefly centrifuged and pellets resuspended in 400 ml cold Buffer A(Hepes pH 7.9 (10 mM), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1mM), PMSF (0.5 mM), pepstatin A (1 mg/ml), leupeptin (10 mg/ml), andaprotinin (10 mg/ml)), on ice for 15 minutes, in the presence of 25 ml1% NP-40. Then, samples were vortexed, centrifuged for 1 minute at10,000 g, and the pellet resuspended with 100 ml Buffer B (Hepes pH 7.9(20 mM), NaCl (400 mM), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (0.5mM), pepstatin A (mg/ml), leupeptin (10 mg/ml) and aprotinin (10mg/ml)). After shaking on a rocker platform for 15 minutes at 4° C.,samples were centrifuged for 15 minutes at 100,000 g at 4° C. 70 mlaliquots were then treated with 150 ml SDS-PAGE sample buffer. Westernblotting was performed as described above, with rabbit anti-mouse NF-kBprimary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) 1:750 inTween TBS (0.02%).

Measurement of nitrite or nitrite/nitrate concentration.

Nitrite in culture supernatants at 24 hours after stimulation wasmeasured as described by Szabo et al., Proc. Natl. Acad. Sci. U.S.A.93:1753-1758 (1996); Zingarelli et al., J. Immunol. 156:350-358 (1996);and Szabo et al., Br. J. Pharmacol. 112:355-356 (1994) by adding 100 mlof Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in5% phosphoric acid) to 100 ml samples of medium. The optical density at550 nm (OD₅₅₀) was measured using a Spectramax 250 microplate reader(Molecular Devices, Sunnyvale, Calif.). For the determination of totalnitrite/nitrate concentrations in plasma samples, nitrate was reduced tonitrite by incubation with nitrate reductase (Zingarelli et al., supra).

Measurement of 6-keto prostaglandin F_(1a).

6-keto prostaglandin F_(1a) production at 4 hours after LPS stimulationwas measured in 100 ml samples of cell culture supernatant using aspecific radioimmunoassay (Szabo et al., Br. J. Pharmacol. 112:355-356(1994)).

Cytokine measurements.

Cytokine levels in plasma and cell culture supernatants were determinedby ELISA. Plasma levels of IL-10 and IL-6 were measured using ELISA kitsfrom Endogen (Endogen Inc., Boston, Mass.). Concentrations of TNF-a inthe plasma and cell culture supernatants were determined using ELISAkits from Genzyme (Genzyme Corp., Boston, Mass.) as described (Szabo etal., Immunology 90:95-100 (1997)).

Measurement of mitochondrial respiration.

Mitochondrial respiration at 24 hours was assessed by themitochondrial-dependent reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan(Szabo et al., Proc. Natl. Acad. Sci. U.S.A. 93:1753-1758 (1996) andZingarelli et al., supra).

Northern blotting for iNOS mRNA.

After exposing cells to LPS in the presence or absence of INH₂BP for 4hours, total RNA was extracted as described using TRIZOL. Aliquotscontaining 15 mg total RNA underwent electrophoresis on a 1% agarose gelcontaining 3% formaldehyde. RNAs were blot transferred to nylon membraneand UV auto-crosslinked. Membranes were hybridized as described byLowenstein et al., Proc. Natl. Acad. Sci. U.S.A. 90:9730-9734 (1993)overnight at 42° C. with a murine iNOS cDNA probe (10⁶ cpm/mL) labeledwith [³²P]-dCTP (specific activity, 3,000 Ci/mM; NEN) by random priming(Pharmacia, Piscataway, N.J.). The hybridized filters were seriallywashed at 53° C. using 2× sodium citrate, sodium chloride, 0.1% SDS and25 mM NaHPO₄, 1 mM EDTA, 0.1% SDS solutions. After probing for iNOS,membranes were stripped with boiling 5 mM EDTA and rehybridized with a[³²P]-radiolabeled ologonucleotide probe for 18S ribosomal RNA as ahousekeeping gene. After washing, exposure was carried out overnightusing a Phosphor Imager screen.

iNOS Western blotting.

Cells were treated with LPS in the presence and absence of pADPRTinhibitor for 20 hours. Cells were then scraped in cold PBS andcentrifuged at 14,000 g for 30 seconds. The supernatant was removed andlysis buffer containing RIPA (500 mL), aprotin (10 mg/ml), and PMSF (0.5mM) was added. DNA was sheered by passing samples through a 22 gaugeneedle. Protein content was determined by the Bradford method (BIO-Rad).Cytosolic protein (200 mg/lane) was added to SDS-PAGE buffer, boiled for5 minutes, separated with 7.5% SDS-PAGE, and transferred tonitrocellulose membranes (0.2 mm) using a Semi-Dry method with anisotachophoretic buffer system. After 1 hour blocking in 3% gelatin andsubsequent washing, the samples were immunoblotted in Tween TrisBuffered Saline (TTBS) and 1% gelatin, with primary rabbit anti-mouseiNOS (upstate Biotechnology, Lake Placid, N.Y.) 1:1000 in TTBS (0.0%)for 2.5 hours. An alkaline phosphatase-conjugated goat anti-rabbit iGGantibody was used as secondary antibody. Antibody binding was visualizedby nitrobule tetrazolium/5-bromo-4-chloro indolyl phosphate (NBT/BCIP)in carbonate buffer (BIO-RAD).

Measurement of iNOS activity.

Cells were treated with LPS in the presence and absence of a pADPRTinhibitor for 12 hours. The measurement of the calcium-independentconversion of L-arginine to L-citrulline in homogenates of the J774cells or in lung homogenates was used as an indicator of iNOS activityas described (Szabo et al., Br. J. Pharmacol. 112:355-356 (1994)). Cellswere scraped or lungs were put into a homogenation buffer composed of 50mM Tris HCl, 0.1 mM EDTA, 0.1 mM EGTA and 1 mM phenylmethylsulfonylfluoride (pH 7.4) and homogenized in the buffer on ice using a TissueTearor 985-370 homogenizer (Biospec Products, Racine, Wis.). Conversionof [³H]-L-arginine to [³H]-L-citrulline was then measured in thehomogenates. Homogenates (30 ml) were incubated in the presence of[³H]-L-arginine (10 mM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM),tetrahydrobiopterin (5 mM) and EGTA (5 mM) for 20 minutes at 22° C.Reactions were stopped by dilution with 0.5 ml of ice cold HEPES buffer(pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Reaction mixtures wereapplied to Dowex 50W (Na+ form) columns and the eluted [³H]-L-citullineactivity was measured by scintillation counting.

Functional assay of iNOS promoter.

J774 cells were resistant to our attempts to transiently transfect themusing the 25 calcium phosphate, lipofectin, and lipofectamin methods,transfection studies were performed in RAW 264.7 cells. iNOS promoteractivity was evaluated by transient transfection of RAW 264.7 cells withreporter gene constructs incorporating the 5′ murine macrophage iNOSpromoter region upstream from the reporter gene luciferase kindlyprovided by Dr. Charles J. Lowenstein, Johns Hopkins University(Lowenstein et al., Proc. Natl. Acad. Sci. U.S.A. 90:9730-9734 (1993)).Two constructs were used: a full length promoter construct (−1592 bp)and a deletional construct consisting of −367 bp. Cells were plated into6-well culture plates at approximately 50% confluence and transfectedwith the respective iNOS promoter-luciferase construct in equimolaramounts using cationic liposomes (Lipofectin, Gibco). In order tocontrol for differences in transfection efficiencies, cells wereco-transfected with pSV40-β-galactosidase. After transfection, cellswere allowed to recover overnight, then subsequently treated with mediaalone (control), LPS (10 mg/ml), or LPS plus INH₂BP (100 mM). After 4hours of treatment, cells were washed once in PBS, lysed in reporterlysis buffer (Promega), and analyzed for luciferase activity.

In vivo experiments.

Male Wistar rats and Male BALB/c mice were obtained from Charles RiverLaboratories (Wilmington, Mass. or Budapest, Hungary). Animals receivedfood and water ad libitum, and lighting was maintained on 12 hour cycle.Rats were injected i.p. with E. coli LPS (15 mg/kg) and sacrificed at 6hours. Plasma samples were taken for nitrite/nitrate determinations andlung samples for iNOS measurements. Separate groups of rats were treatedwith INH₂BP (10 mg/kg i.p.) 10 minutes prior to LPS or 2 hours after LPSinjection.

In studies for the measurement of LPS-induced cytokine response, micewere injected i.p. with either drug vehicle, or with INH₂BP (10 mg/kg)in a volume of 0.1 ml/10 g body weight. Half an hour later they werechallenged with 4 mg/kg of i.p. LPS. The animals were killed at 90minutes after LPS treatment, blood was collected in ice-cold Eppendorftubes containing EDTA, and centrifuged for 10 minutes at 4° C. Theplasma was stored at −7° C. until assayed.

In survival studies with mice, animals were subjected to i.p. injectionof LPS (120 mg/kg) at time 0, and survival was monitored for 42 hoursafter LPS. Separate groups of mice received vehicle or INH₂BP treatment(0.1-10 mg/kg i.p.) at times −18 hours, −4 hours, 0 hours, 6 hours, 24hours and 30 hours relative to LPS.

Materials.

DMEM, RPM1, TRIZOL and fetal calf serum were from Gibco (Grand Island,N.Y.). [³H]-NAD⁺ and [³²P]-NAD⁺ were obtained from DuPont NEN (Boston,Mass.). Alcohol dehydrogenase and ND⁺ were obtained from BoehringerMannheim (Indianapolis, Ind.). PD 98059 was obtained from Cal Biochem(La Jolla, Calif.). All other drugs were obtained from Sigma (St. Louis,Mo.).

Statistical evaluation.

All values in the figures and text are expressed as mean ±standard errorof the mean (S.E.M.) of n observations (n≧4). Student's unpaired t-testwas used to compare means between groups. A p-value less than 0.05 wasconsidered statistically significant.

Results

INH₂BP suppresses LPS-induced nitric oxide and prostaglandin but notTNF-α production in J774 macrophages

INH₂BP treatment caused a dose-dependent inhibition of LPS-inducednitrite formation in J774 macrophages (FIG. 1a). Similarly, INH₂BPsuppressed LPS-induced production of 6-keto prostaglandin F_(1a) (FIG.1b), but not the production of TNF (FIG. 1c), and restored theLPS-induced suppression of mitochondrial respiration (FIG. 1d). INH₂BPcaused a marked inhibition of iNOS mRNA and protein expression (FIGS.2a-c). The inhibition of nitrite production by INH₂BP was greatlydiminished when the agent was given several hours LPS, as opposed toprior to the stimulus of iNOS induction (FIG. 3a). Moreover, theinhibitory effect of INH₂BP on iNOS was greatly reduced when LPS wasused in combination was with interferon-gamma (INF-g 50 u/mL) forimmunostimulation (FIG. 3b).

Selective regulation of the induction of the iNOS promoter by INH₂BP

In order to further study the regulation of iNOS promoter by INH₂BP weperformed transient assays using murine macrophage iNOSpromoter-luciferase constructs. Consistent with previous data(Lowenstein et al., supra), we found an important role for LPS-mediatedtranscriptional regulation of murine macrophage iNOS, as evidenced by a10 to 12-fold induction of luciferase activity by LPS (FIG. 4).Co-treatment of cells transfected with the full length (−1592 bp)promoter construct with INH₂BP, completely inhibited LPS-mediatedluciferase activity (FIG. 4). However, similar co-treatment of cellstransfected with the −367 bp deletional construct did not significantlyaffect LPS-mediated luciferase activity (FIG. 4).

In vivo antiinflammatory effects of INH₂BP

INH₂BP pretreatment significantly reduced the LPS-induced increase inplasma nitrite-nitrate and the increase in pulmonary iNOS activity inconscious rats (FIG. 5). The inhibitory effect of INH₂BP on NOproduction was reduced when the agent was added to the cells or to theanimals several hours after LPS stimulation (FIG. 5). Similarly to thetransformed cell lines, treatment with 100 mM INH₂BP significantlyreduced (by 56±7%, p<0.01) nitrite production in primary cells(peritoneal macrophages obtained from rats) stimulated with LPS (10mg/ml) in vitro (n=4).

Similarly to the in vitro results (FIG. 1c), INH₂BP did notsignificantly affect the LPS-induced increase in plasma TNF levels inmice (FIG. 6a) nor did INH₂BP affect LPS-induced IL-6 production (FIG.6C). However, INH₂BP caused an augmentation of the LPS-induced IL-10plasma response (FIG. 6b).

Pretreatment of mice by INH₂BP caused a significant and dose-dependentimprovement in the survival rate subjected to lethal doses of LPS (FIG.7).

INH₂BP suppresses the LPS-induced induction of iNOS.

The inhibitory effect of INH₂BP on iNOS expression was indicated by theinhibition on nitrite production, iNOS mRNA expression and iNOS proteinexpression. The regulation occurs in the early stage of iNOS induction,since INH₂BP gradually loses its effectiveness when applied atincreasing times after the stimulus for iNOS induction. The regulationof INH₂BP of iNOS induction occurs both in vitro and in whole animals.In addition, our data show that the LPS-induced production ofcyclooxygenase metabolites, similar to the induction of iNOS, ismodulated by INH₂BP. The production of cyclooxygenase metabolites bypro-inflammatory cytokines is due to novel mRNA and protein synthesis,and expression of COX-2, by a process which shares similarities with theprocess of iNOS induction (Vane et al., Inflamm. Res. 44:1-10 (1995)).The inhibition of the LPS-induced expression of inflammatory mediators,however, is not a non-specific response to INH₂BP, since the inductionof TNF by LPS was not affected by this agent in the J774 cells.

Interestingly, the inhibitory effect of INH₂BP on iNOS was greatlyreduced when LPS was used in combination with INF for immunostimulation.This effect may be due to the fact that IFN-induced transcriptionfactors such as interferon-regulatory factor (Martin et al., J. Exp.Med. 180:977-84 (1994)) bypass the inhibition of the iNOS induction bythe above mentioned agents.

Previous in vitro studies have suggested that induction of iNOS ismodulated by pharmacological inhibitors of pADPRT in macrophages invitro (Hauschildt et al., Biochem. J. 288:255-260 (1992) andPellat-Seceunyk et al., Biochem. J. 297:53-58 (1994)). However, in thesestudies, the pADPRT inhibitors aminobenzamide and nicotinamide were usedat high concentrations (10-30 mM), which inhibited total protein and RNAsynthesis, and may have had additional, pharmacological actions, such asfree radical scavenging (Hauschildt et al., supra).

The present experiments, using INH₂BP, further suggest the pleiotropicinvolvement of pADPRT in the process of iNOS mRNA transcription. Inorder to study the regulation of the iNOS promoter by INH₂BP, transienttransfection assays were performed using murine macrophage iNOS promoterluciferase constructs. These data with the deletional constructsindirectly suggest that INH₂BP regulates a transcription event whichinvolves the murine iNOS promoter region between −1592 and −367 bp.ADP-ribosylation of histones and nucleases may be involved in themaintenance of a relaxed chromatin structure (Bauer et al., Int. J.Oncol. 8:239-252 (1995), Bauer et al., Biochimie 77:347-377 (1995), andUeda et al., Ann. Rev. Biochem. 54:73-100 (1985)). Based on previousexperimental data, it is reasonable to suggest that in theseexperimental systems pADPRT inhibitory compounds, e.g., INH₂BP,pretreatment inhibits auto-poly-ADP-ribosylation of pADPRT and histones.Such action is known to trigger the conversion of relaxed to condensedchromatin, and, by way of upregulation of nucleases and other DNAstructure regulatory enzymes may affect promoter functions.

Pathophysiological and therapeutic implications; INH2BP modulates theinflammatory process at multiple levels.

Reduction by pADPRT inhibitors of the expression of pro-inflammatorygenes iNOS and COX-2, and the subsequent reduced formation of NO andprostaglandins may be beneficial in various forms of inflammation. Inaddition, enhanced release of IL-10 may have additionalanti-inflammatory actions (Liles et al., J. Infect Dis. 172:1573-80(1995), Giroir, Critical Car. Med. 21:780-9 (1993) and Szabo et al.,Immunology 90:95-100 (1997)). It is conceivable that such effectssignificantly contribute to the improvement by pADPRT inhibitorycompounds, e.g., INH₂BP pretreatment and the survival rate of micechallenged with lethal doses of endotoxin.

On one hand, it is conceivable that pADPRT activity or the binding ofpADPRT protein is involved in the regulation of the production ofinflammatory mediators and/or the expression of genes that code forcomponents of the inflammatory process. On the other hand, it isprobable that indirect down-regulation of MAP kinase activity by INH₂BP(Bauer et al., Int. J. Oncol. 8:239-252 (1995)) may also contribute tothe observed effects, as predicted by other studies (Kyriakis et al., J.Biol. Chem. 271:24313-24316 (1996) and Ferrell, TIBS 21:460-466 (1996)).The present results demonstrate the therapeutic potential of pADPRTinhibitory compounds such as INH₂BP in various inflammatory diseases.

EXAMPLE 2

Induction and evaluation of collagen-induced arthritis

Male DBA/1J mice (9 weeks, Jackson Laboratory, Bar Harbor, Me.) wereused for these studies. Chick type II collagen (CII) was dissolved in0.01 M acetic acid at a concentration of 2 mg/ml by stirring overnightat 4° C. Dissolved CII was frozen at −70° C. until use. CompleteFreund's adjuvant (CFA) was prepared by the addition of Mycobacteriumtuberculosis H37ra at a concentration of 2 mg/ml. Before injection, CIIwas emulsified with an equal volume of CFA. Collagen-induced arthritiswas induced as previously described by Hughes et al., J. Immunol.153:3319-3325 (1994). On day 1, mice were injected intradermally at thebase of the tail with 100 mL CII. On day 21, a second injection of CIIin CFA was administered. Animals were treated with either vehicle (n=10)or with INH₂BP (n=60(0.5 g/kg p.o.) every 24 hours, starting from Day25. Mice were evaluated daily for arthritis by using a macroscopicscoring system ranging from 0 to 4 (1—swelling and/or redness of the pawor one digit; 2—two joints involved; 3—more than two joints involved;and 4—severe arthritis of the entire paw and digits). The arthriticindex for each mouse was calculated by adding the four scores of theindividual paws. At the end of the experiments (Day 35), animals weresacrificed under anesthesia, and paws and knees were removed and fixedfor histological examination. Histological examination was performed byan investigator blinding for the treatment regime.

Data analysis and presentation

For the studies with carrageenan-induced paw edema, paw volumes in thetreated and untreated groups of animals were compared with unpairedStudent's test. For the arthritis studies, Mann-Whitney U-test(2-tailed, independent) was used to test the statistical differences inthe arthritic indices. This nonparametric statistic was used to comparemedians, rather than means, because the scale of measurement wasordinal, and the distribution values were typically nonnormallydistributed; Hughes et al., supra.

Values in FIG. 10 are expressed as mean ±standard error of the mean of nobservations, where n represents the number of rats (6 animals for eachgroup). Values in FIG. 11 represent incidences (%), whereas values inFIG. 12 represent medians. A P-value less than 0.05 was consideredstatistically significant (I′<0.05; **p<0.02).

Materials

5-iodo-6-amino-1,2-benzopyrone (INH₂BP) was prepared as described inExample 1. Chick type II collagen was obtained from Elastin ProductsCompany, Inc. (Owensville, Mo.). Mycobacterium tuberculosis H37Ra wasfrom Difco (Detroit, Mich.). All other chemicals were from SigmaChemical Co. (St. Louis, Mo.). Subplantar injection of carrageenan intothe rat paw led to a time-dependent increase in paw volume with amaximal response at 3 hour (FIG. 10). This carrageenan induced paw edemawas significantly reduced by treatment with INH₂BP (FIG. 10).

In the collagen-induced arthritis model in mice, between Days 26-35after the first collagen immunization, animals progressively developedarthritis, as evidenced by an increase in the arthritis incidence and anincrease in the arthritic score (FIGS. 6-7). Treatment with INH₂BPreduced the incidence of arthritis until Day 33 and reduced the severityof the disease throughout the experimental period. By Day 30, arthriticscore increased to 10, whereas median arthritic scores in the INH₂BPtreated animals remained around 5 (FIG. 12). By Day 35, allvehicle-treated animals, and most of the INH₂BP treated animals had somedegree of arthritis (FIG. 11). However, even at Day 35, the medianarthritic scores were significantly decreased by INH₂BP treatment (FIG.12).

At Day 35, histological evaluation of the paws in the vehicle-treatedarthritic animals revealed signs of severe suppurative arthritis, withmassive mixed (neutrophil, macrophages and lymphocyte) infiltration intothe larger ankle joints and the terminal digits. In addition, a severeor moderate necrosis, hyperplasia and sloughing of the synovium could beseen, together with the extension of the inflammation into the adjacentmusculature with fibrosis and increased mucous production. In theanimals treated with INH₂BP, the degree of arthritis was significantlyreduced. Nevertheless, there was still a significant degree of arthritisin these animals, with a moderate, primarily neutrophil infiltrationinto several of the larger joints, coupled with mild to moderatenecrosis and hyperplasia of the synovium. Similar to these findings inthe paw, signs of severe suppurative arthritis were found in the knee,which was reduced by treatment with INH₂BP (not shown).

Discussion

NO, peroxynitrite, oxyradicals and products of the induciblecyclooxygenase have independently been proposed as important factors inthe pathogenesis of various forms of inflammation, including arthritis(Brahn, Clin. Orthop. Rel. Res. 265:42-53 (1991); Kaur et al., FEBSLett. 1359:9-12 (1994); Oyanagui Y, Life Sci. 54:PL285-9 (1994); Mieselet al., Inflammation 6:597-612 (1994); Whiteman et al., Annals. of theRheumatic Diseases 55:383-7 (1996); Anderson et al., J. Clin. Invest.97:2672-2679 (1996)). The present study, demonstrating anti-inflammatoryeffects of INH₂BP in the carrageenan-induced paw edema model and in thecollagen induced arthritis model supports the view that PARS is involvedin the progression of the inflammatory process and the pharmacologicalinhibition of PARS is of anti-inflammatory potential.

The overproduction of NO in inflammatory conditions is due to thesuppression of the inducible isoform of NOS (iNOS). Several lines ofevidence suggest a role for iNOS and NO overproduction in thepathogenesis of arthritis (Stenovic-Racic, et al., Arthr. Rhemat.36:1036-1044 (1993)). First, the expression of iNOS and the productionof large amounts of NO has been demonstrated in chondrocytes fromexperimental animals and humans (Haeselmann et al., FEBS Lett.352:361-364 (1994); Sakurai et al., J. Clin. Invest. 96:2357-63 (1995);Grabowski et al., Br. J. Rheumatol. 35:207-12 (1996); Murrell et al., J.Bone Joint Sur.—Am. 78:265-74 (1996). Second, an increase in thecirculating levels of nitrite/nitrate (the breakdown products of NO) hasbeen demonstrated in patients with arthritis (Farrell et al., Ann. Rhem.Dis. 51:1219-22 (1992); Stichtenoth, et al., Ann. Rhem. Dis. 54:820-4(1995). Third, the development of arthritis has been shown to be reducedby non-isoform-selective inhibitors of NOS (McCartney-Francis et al., J.Exp. Med. 178:749-753 (1993); Weinberg et al., J. Exp. Med. 1979:651-60(1994); Stefanovic-Racic et al., Arthr. Rheumat. 37:1062-9 (1994); and,more recently, by inhibitors with selectivity for iNOS (Connor et al.,Eur. J. Pharmacol. 273:15-24 (1995).

In this respect it is noteworthy that pretreatment of multiple celltypes with PARS inhibitors (including 3-aminobenzaminde, nicotinamide aswell as INH₂BP) prior to immunostimulation has been shown to suppressthe expression of mRNA for iNOS and reduce NO production (Zingarelli etal J. Immunol. 156:350-358 (1996)). From these experimental data it maybe concluded that PARS via a not yet characterized mechanism, alsoregulates the process of iNOS expression, and that this effect mayrepresent an additional mode of beneficial action of PARS inhibition invarious forms of inflammation. In the in vitro studies cited above,extremely high concentrations of the PARS inhibitors 3 -amiobenzamideand nicotinamide were required (10-30 mM) in order to demonstratesuppression of iNOS induction. These high concentrations of these agentsmay have additional pharmacological actions, such as inhibition of totalprotein and RNA synthesis, and/or free radical scavenging actions(Zingarelli et al., supra). INH₂BP, on the other hand, effectivelysuppressed the expression of iNOS even at lower, non-cytotoxicconcentrations (100-300 mM).

EXAMPLE 3

Protection against peroxynitrite-induced fibroblast injury and arthritisdevelopment by inhibition of poly (ADP-ribose) synthetase

Cell culture

Mouse embryo fibroblasts from a PARS^(−/−) mouse (a geneticallyengineered mouse that lacks the gene for PARS) and fibroblasts from acorresponding wild-type control (Wang et al., (1995) Genes Develop. 9:510-520) were grown in Dulbecco's modified Eagle's Medium with 10% fetalbovine serum. Cells were cultured in 96-well plates or in 12-well platesuntil 90% confluence. Cells were exposed to peroxynitrite (25-1000 μM)in the presence or absence of a 10 min pretreatment with INH₂BP (100μM). For immunostimulation, cells were exposed to bacteriallipopolysaccharide (LPS, 10 μg/ml) and murine gamma-interferon (IFN, 50U/ml) for 2-48 hours in the presence or absence of INH₂BP (50-100 μM).INH₂BP was synthesized as described in Example 1.

Determination of DNA single strand breaks and measurement of cellularPARS activity

At 10 min after peroxynitrite exposure, the formation of DNA singlestrand breaks in double-stranded DNA was determined by the alkalineunwinding method as previously described by Szabó, C. (1996) Shock 6:79-88; Szabó et al., (1996) Proc. Natl. Acad. Sci. USA 93: 1753-1758.PARS activity was measured 10 min after peroxynitrite exposure, usingradiolabeled NAD⁺ as described, in digitonin-permeabilized cells.

Measurement of mitochondrial respiration and cellular NAD+ levels.

At 60 min after peroxynitrite exposure or 48 h after immunostimulation,respiration was assessed by the mitochondrial-dependent reduction of MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] toformazan as described by Szabó et al., supra, referenced in Example 1.In addition, in some experiments, cellular NAD⁺ levels were determinedusing HPLC.

Measurement of nitrite or nitrite/nitrate production, iNOS mRNA and iNOSprotein expression by immunostimulated cells.

Nitrite in culture supernatants at 24 hours after stimulation wasmeasured by the Griess reaction as described by Szabó et al., Circ. Res.78:1051-1063 (1996). For the determination of total nitrite/nitrateconcentrations, nitrate was reduced to nitrite by incubation withnitrate reductase. After exposing cells to LPS/IFN in the presence orabsence of INH₂BP (100 μM) for 1-24 hours, Northern blotting for iNOSmRNA and Western blotting for iNOS protein, using a primary rabbitanti-mouse iNOS antibody (Upstate Biotechnology, Lake Placid, N.Y.) wereperformed as described (Zingarelli et al., J. Immunol. 156:350-358(1996)). The activity of iNOS in cell homogenates was determined by themeasurement of the calcium-independent conversion of L-arginine toL-citrulline.

Effect of INH₂BP on peroxynitrite- and hydrogen peroxide-inducedoxidation of dihydrorhodamine 123.

The effect of INH₂BP and the conventional PARS inhibitor3-aminobenzamide (100 μM-3 mM) on the peroxynitrite-mediated oxidationof dihydrorhodamine 123 was studied in vitro. These studies wereperformed in phosphate-buffered saline (PBS) containing 100 μMdiethylenepentaacetic acid (DTPA), pH 7.4. The oxidation of DHR 123 byperoxynitrite (1 μM) or hydrogen peroxide (1 μM) plus 25 μg/mlhorseradish peroxidase in the presence of various concentrations of thePARS inhibitors was measured by the change in absorbance at 500 nm(γ=78,000 M⁻¹ cm⁻¹) after 30 min incubation at 37° C.

Induction of collagen-induced arthritis, and detection of nitrotyrosinein the inflamed joints

Male DBA/1J mice (9 weeks, Jackson Laboratory, Bar Harbor, Me.) wereused for these studies. Chick type II collagen (CII) was dissolved in0.01 M acetic acid at a concentration of 2 mg/ml by stirring overnightat 4° C. Dissolved CII was frozen at −70° C. until use. CompleteFreund's adjuvant (CFA) was prepared by the addition of Mycobacteriumtuberculosis H37Ra at a concentration of 2 mg/ml. Before injection, CIIwas emulsified with an equal volume of CFA. Collagen-induced arthritiswas induced as previously described (Hughes et al., J. Immunol. 153:3319-3325 (1994)). On day 1, mice were injected intradermally at thebase of the tail with 100 μl of the emulsion (containing 100 μg CII). Onday 21, a second injection of CII in CFA was administered. At Day 35,joints were taken, embedded in M1 medium and snap frozen in liquidnitrogen. Cryostat sections (6 μm) were cut with a microtome equippedwith a carbide steel knife. Joint sections were analyzed for thepresence of nitrotyrosine, an indicator of peroxynitrite byimmunohistochemistry as described, using a primary anti-nitrotyrosineantibody (Upstate Biotech, Saranac Lake, N.Y.). In control experiment,sections were incubated in the presence of 10 mM nitrotyrosine. Thisintervention eliminated the nitrotyrosine staining presented in FIG. 16.

In another set of studies, aqueous joint extracts were prepared fromcontrol animals and from animals at 35 days of arthritis as described byKasama et al., J. Clin. Invest. 95: 2868-2876 (1995), by homogenizationin a lysis buffer in the presence of protease inhibitors. Extracts wereanalyzed for the presence of nitrated proteins, using Western blotting,as described by Cuzzocrea et al., Br. J. Pharmacol. 122: 493-503 (1997).

Induction of collagen-induced arthritis, and its suppression by INH₂BP

In another set of experiments, PARS was inhibited in the animals withINH₂BP. Animals were treated with either vehicle (n=10) or with INH₂BP(n=6) (0.5 g/kg p.o.) every 24 hours, starting from Day 25. Experimentswith ¹⁴C-labeled INH₂BP have established that the dosage regimen used inthis study provides adequate tissue uptake of the PARS inhibitor (Baueret al., Int. J. Oncol. 8: 239-252 (1996)). Mice were evaluated daily forarthritis by using a macroscopic scoring system: 0=no signs ofarthritis; 1=swelling and/or redness of the paw or one digit; 2=twojoints involved; 3=more than two joints involved; and 4=severe arthritisof the entire paw and digits. Id. Arthritic index for each mouse wascalculated by adding the four scores of individual paws. At Day 35,animals were sacrificed under anesthesia, and paws and knees wereremoved and fixed for histological examination, which was done by aninvestigator blinded for the treatment regime.

Data analysis and presentation.

For the in vitro studies, all values in the figures and text areexpressed as mean ±standard error of the mean of n observations, where nrepresents the number of wells studied (6-9 wells from 2-3 independentexperiments). Data sets were examined by one-and two-way analysis ofvariance and individual group means were then compared with Student'sunpaired t-test. For the arthritis studies, Mann-Whitney U-test(2-tailed, independent) was used to compare medians of the arthriticindices. Values in for the in vitro studies are presented as incidences(%), or medians. A p-value less than 0.05 was considered statisticallysignificant.

Results

Role of PARS activation in the peroxynitrite-mediated inhibition ofmitochondrial respiration in fibroblasts.

Exposure of wild-type (PARS^(+/+)) fibroblasts to peroxynitrite (50-1000μM) caused a dose-dependent suppression of the mitochondrial respirationat 1 h (FIG. 13a). In addition, peroxynitrite dose-dependently increasedthe percentage of single-strand breaks of the DNA in these cells. Forinstance, at 100 μM peroxynitrite, the percentage of single strandbreaks increased from 3±2% to 28±2% (p<0.01) (n=6). Peroxynitrite alsocaused a dose-dependent activation of PARS (FIG. 13b), with some basalPARS activity detectable in unstimulated wild-type cells (FIG. 13b).INH₂BP prevented PARS activation in response to peroxynitrite (FIG.13b), without affecting the extent of DNA single strand breakage (notshown). Pharmacological inhibition of PARS caused a significantprotection against the peroxynitrite-induced suppression ofmitochondrial respiration at 50 μM, 100 μM (FIG. 13a) and 250 μM (notshown) peroxynitrite. However, when cells were exposed to very highconcentrations of peroxynitrite (1000 μM), the suppression ofmitochondrial respiration could no longer be prevented bypharmacological inhibition of PARS, indicating non-specific cellulardamage (FIG. 13a). The extent of DNA single strand breakage in thePARS^(−/−) cells was similar to the DNA single strand breakage in thePARS^(+/+) controls. For instance, at 100 μM peroxynitrite, thepercentage of single strand breaks increased from 3±2% to 31±4% in thesecells (p<0.01) (n=6). When the cellular responses in the fibroblast linederived from the PARS^(−/−) mice were compared to the response in thecorresponding wild-type cells, the results were similar to what we haveobserved with the pharmacological inhibitor, INH₂BP. Cells from thePARS^(−/−) mice were protected against peroxynitrite-induced suppressionof mitochondrial respiration (without adding INH₂BP) (FIG. 13). Thisprotection diminished when extremely high peroxynitrite concentrationswere used (e.g. 1000 μM). The PARS^(−/−) cells were also protectedagainst the peroxynitrite-induced suppression of cellular NAD⁺ levels.For instance, 100 μM peroxynitrite caused a complete depletion ofcellular NAD⁺ in the wild-type cells from (8.2±1.6 to 0.1±0.1 nmoles/mgprotein; p<0.01, n=3); whereas NAD⁺ was well maintained in thePARS^(−/−) cells (control: 9.9±0.5 nmoles/mg protein; after 100 μMperoxynitrite exposure: 5.0±0.9 nmoles/mg protein). Even at very highconcentrations of peroxynitrite, where no protection against thesuppression of mitochondrial respiration was provided in the PARS^(−/−)phenotype, cellular NAD⁺ levels in the PARS^(−/−) cells were relativelymaintained. For instance, in response to 1000 μM peroxynitrite, cellularNAD⁺ was 5.1±0.9 nmoles/mg protein in the PARS^(−/−) cells (n=3). Theseresults demonstrate that PARS activation plays an important role in thecellular injury at low to intermediate concentrations of peroxynitrite.However, at extremely high oxidant concentrations, overwhelmingPARS-independent mechanisms of cytotoxicity become activated. Thislatter finding is in accordance with observations in pancreatic isletcells, macrophages and endothelial cells, where extremely highconcentrations of oxidants caused massive cytotoxicity, which was nolonger preventable by pharmacological inhibition of PARS (Szabo et al.,Proc. Natl. Acad. Sci. USA 93:1753-1758 (1996); Szabo et al., FEBS Lett.372:229-232 (1995); Kasama et al., J. Clin. Invest. 95:2868-2876(1995)).

In order to directly investigate any potential scavenging effect ofINH₂BP, in vitro studies were performed with INH₂BP, and3-aminobenzamide, a prototypical PARS inhibitor, in an assay whichutilizes the peroxynitrite- or hydrogen peroxide induced oxidation ofdihydrorhodamine 123. The results showed that INH₂BP does not inhibitthe peroxynitrite- or hydrogen peroxide induced oxidation, whereas inline with previous studies, 3-aminobenzamide dose-dependently inhibitedthe oxidation of dihydrorhodamine induced by hydrogen peroxide, but notby peroxynitrite (FIG. 14). These observations, coupled with the findingthat in the PARS^(−/−) cells, which resisted the suppression ofmitochondrial respiration at low to intermediate concentrations ofperoxynitrite, INH₂BP did not provide any non-specific additionalprotection (FIG. 13a), indicate that INH₂BP does not act as a scavengerof peroxynitrite.

Role of PARS in the regulation of NO production in response toimmunostimulation.

Stimulation of the cells with LPS and interferon-gamma induced theproduction of nitrite and nitrate (breakdown products of NO, produced bythe inducible NO synthase enzyme, iNOS) in the fibroblasts, as measuredat 24 hours. There was a significantly lower nitrite and nitrateproduction in response to immunostimulation in the PARS^(−/−) cells,when compared to wild-type controls. INH₂BP (50-100 μM) caused adose-dependent inhibition of nitrite and nitrate production in thewild-type cells, lowering it to the level found in the PARS^(−/−) cells(FIG. 15a). INH₂BP, however, did not inhibit NO production in thePARS^(−/−) cells (FIG. 15a). Similar differences in the NO productionpersisted at 48 hours after immunostimulation.

In the PARS^(−/−) cells, there was a significantly lower expression ofiNOS, as indicated by lower amounts of iNOS steady-state mRNA and iNOSprotein levels (FIG. 15 b). The mRNA for iNOS was 2.8±0.7 fold higher at8 hours after immunostimulation and 4.6±1.9 fold higher at 24 hoursafter immunostimulation in the PARS^(+/+) cells, than in the PARS^(+/+)cells (n=3; p<0.01). Direct measurements of iNOS activity inimmunostimulated cells confirmed these results: at 12 hours afterimmunostimulation, calcium-independent iNOS activity amounted to 306±39nmoles/mg/min in PARS^(+/+) cells, 95±11 nmoles/mg/min in INH₂BPpretreated PARS^(+/+) cells, 90±24 nmoles/mg/min in PARS^(−/−) cells and76±38 nmoles/mg/min in INH₂BP pretreated PARS^(−/−) cells (n=6). Thelack of effect of INH₂BP in the PARS^(−/−) cells reiterates that thisagent does not exert PARS-independent cellular actions.

At 48 hours after immunostimulation, changes in mitochondrialrespiration in PARS^(+/+) and PARS^(−/−) cells were also compared. Therewas a 46±6% inhibition of the respiration in response to LPS/IFN in thePARS^(+/+) cells (p<0.01, n=12), whereas in the PARS^(−/−) cells, nosuppression of mitochondrial respiration was observed: the respirationamounted to 114±8% of the unstimulated controls (n=12, p<0.01).

Effect of INH₂BP in the development of collagen-induced arthritis.

NOS inhibitors and superoxide dismutase mimics exert protective effectsin rodent models of arthritis, induced by adjuvant (McCartney-Francis etal., J. Exp. Med. 178:749-753; Stefanovic-Racic et al., Arthr. Rheumat.37:1062-1069 (1994)) or collagen (Brahn et al., FASEB J. 11:A530(1997)). Using immunohistochemistry, and Western blotting of proteins inaqueous joint extracts, we observed the appearance ofnitrotyrosine-positive staining in the inflamed joints, but not inhealthy animals (FIG. 16). These findings are in accordance with arecent study in human samples from arthritic patients (Kaur et al., FEBSLett. 350:9-12 (1994)). Nitrotyrosine formation is generally accepted asa specific “footprint” of peroxynitrite (Beckman et al., Am. J. Physiol.271:C1424-1437 (1996); Ischiropoulos et al., Arch. Biochem. Biophys.298:431-437 (1992)), although recent studies proposed additionalpathways of tyrosine nitration, such as the one related to themyeloperoxidase-dependent conversion of nitrite to NO₂Cl and NO₂(Eiserich et al., Nature, in press). Thus, nitrotyrosine may ratherserve as a collective indicator of “reactive nitrogen species”(Halliwell, FEBS Lett. 411:157-160 (1997)).

Based on our in vitro data indicating the importance of theperoxynitrite-PARS pathway in cell injury, we used INH₂BP to define therole of PARS in a mouse model of collagen-induced arthritis. BetweenDays 26-35 after the first collagen immunization, animals progressivelydeveloped arthritis (FIG. 17a-b). INH₂BP reduced the incidence ofarthritis until Day 33 and reduced the severity of the diseasethroughout the experimental period. At Day 35, histological evaluationof the paws in the vehicle-treated arthritic animals revealed signs ofsevere suppurative arthritis, with massive mixed (neutrophil, macrophageand lymphocyte) infiltration. In addition, severe or moderate necrosis,hyperplasia and sloughing of the synovium could be seen, together withthe extension of the inflammation into the adjacent musculature withfibrosis and increased mucous production (FIG. 18b). In the INH₂BPtreated animals, the degree of arthritis was significantly reduced: amoderate, primarily neutrophil infiltration into several of the largerjoints, coupled with mild to moderate necrosis and hyperplasia of thesynovium (FIG. 18c).

EXAMPLE 4

Here we demonstrate that inhibition of PARS with the novel, potent PARSinhibitor 5-iodo-6-amino-1,2-benzopyrone (INH₂BP) protects againstperoxynitrite-induced cell death in C6 glioma cells in vitro, andprotects against the development of infarct and neurological deficit ina murine stroke model.

Methods

In vitro studies

The rat astrocytoma cell line C6 was cultured in Ham's F12 medium with15% horse serum and 2.5% fetal calf serum. After a 10 min INH₂BP (1-100μM) or vehicle pretreatment, cells were stimulated with peroxynitrite(500 μM) for 20 min (PARS assay) or 1 h (MTT and LDH assay) at 37° C.The incorporation of tritiated NAD⁺ into nuclear proteins, an index ofPARS activation was measured, as described by Szabó et al., J. Clin.Invest. 100:723-735 (1997). Reduction of[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), anindicator of mitochondrial respiration, was measured, and cell membraneinjury was quantitatively assessed by the measurement of lactatedehydrogenase (LDH) release. Id.

In vivo studies.

Adult male 129/SV mice were anesthetized with halothane. A 2 hourischemia, followed by a 22 hour reperfusion, was induced with a 8.0nylon monofilament coated with silicone resin/hardener mixture asdescribed by Hara et al., J. Cereb. Blood Flow Metab. 16: 605-611(1996). INH₂BP 10 mg/kg or 30 mg/kg in 5% dimethylsulfoxide (DMSO) inphosphate buffered saline (PBS, pH 7.4) was administered in a volume of0.3 mL i.p. 2 hours before induction of ischemia. Control animals wereinjected i.p. with a corresponding volume of 5% DMSO in PBS. Infarctmeasurements were performed in five coronal 2 mm-sections with an imageanalysis system (Huang et al., Science 265:1883-1885 (1994)).Neurological deficit was also assessed by a 1-4 scoring system by Haraet al., supra.

Data analysis.

Data are presented as mean ±standard error (SE). Comparisons were madeby two-tailed student's t-test. For neurological deficits,Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Dunn'stest was used. P<0.05 was considered statistically significant.

Results

INH₂BP inhibits peroxynitrite-induced PARS activation and neuronalinjury in glial cells in vitro.

Pretreatment with INH₂BP (1-100 μM) caused a dose-dependent inhibitionof the peroxynitrite induced activation of PARS in the C6 cells. Themost potent effect of INH₂BP was achieved with 100 μM concentration(FIG. 19a). Peroxynitrite induced a decrease in MTT reduction and anincrease in the LDH levels in the culture medium, indicative ofsuppressed mitochondrial respiration and disrupted cell membraneintegrity, respectively (FIG. 19a). INH₂BP dose-dependently protectedagainst peroxynitrite-induced injury (FIG. 19a).

INH₂BP reduces infarct size after transient middle cerebral arteryocclusion.

Pretreatment with 30 mg/kg i.p. (n=8) 2 hours before ischemiasignificantly reduced infarct size after 2 hours of middle cerebralartery occlusion and reperfusion in 129/SV mice compared to controls(n=10) (FIG. 19b). With INH₂BP at 10 mg/kg (n=11), there was a trend tosmaller infarct sizes compared to controls (FIG. 19b). All animalsexhibited a neurological score of 2 or higher 30 minutes after the onsetof ischemia. At 22 hours reperfusion, deficits were significantly(p<0.05) improved in the 30 mg/kg group compared with controls (1.8±0.1vs 1.2±0.3 vs 1.0±0.2 in vehicle-treated, 10 and 30 mg/kg INH₂BP-treatedanimals, respectively).

Discussion

These data demonstrate that inhibition of PARS with INH₂BP provides adose-dependent protection against glial cell injury in vitro and againststroke development in vivo. Along with a reduction in infarct size,neurological deficit was improved in treated animals, indicatingfunctional recovery after PARS inhibition.

A significant portion of the neuronal injury is related tooverproduction of NO, due to the N-methyl-D-aspartate receptoractivation, and subsequent activation of the neuronal NO synthase in thereperfused brain. NO combines with superoxide, peroxynitrite, in turn,induces rapid and pronounced oxidative and peroxidative injury (Dalkaraet al., Int. Rev. Neurobiol. 40: 319-336 (1997)). Part of this injury isrelated to DNA single strand breakage and activation of anenergy-consuming cycle by PARS. The data provided herein demonstratethat the experimental therapy of stroke may represent a novel indicationfor the development of INH₂BP and related PARS inhibitors.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described modes for carrying out theinvention which are obvious to those skilled in the field ofpharmaceutical formulation or related fields are intended to be withinthe scope of the following claims.

What is claimed is:
 1. A method for treating arthritis in an animal ormammal, said method comprising the step of administering to an animal ormammal a therapeutically effective amount of an pADPRT inhibitorycompound, wherein said pADPRT inhibitory compound is not a benzamide. 2.The method of claim 1 wherein the compound is selected from the groupconsisting of: a compound having the structural formula:

 wherein R₁, R₂, R₃, R₄, R₅ and R₆ are, independent of one another,selected from the group consisting of hydrogen, hydroxy, amino, nitroso,nitro, halogen, (C₁-C₆) alkyl, (C₁-C₆) alkoxy, (C₃-C₇) cycloalkyl, andphenyl and pharmaceutically acceptable salts thereof, wherein at leastthree of the six R₁, R₂, R₃, R₄, R₅ and R₆ substituents are alwayshydrogen and at least one of the six R₁, R₂, R₃, R₄, R₅ and R₆substituents are selected from the group consisting of amino, nitrosoand nitro.